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The epidermal growth factor receptor (EGFR) is a therapeutic target in a number of settings in solid malignancies, but its role in breast cancer has remained unclear and controversial. In 810 primary breast cancers derived from patients suitable for cytotoxic chemotherapy, EGFR was prospectively measured and interactions with tumour and clinical correlates were tested to observe whether postulated cross-talk mechanisms are likely to modulate breast cancer metastasis and proliferation. A minority (79 tumours, 9.8%) were EGFR positive; in a multivariate analysis the likelihood of being EGFR positive was significantly increased for patients with grade 3 disease, compared with grade 1 (OR 15.6; 95% CI 2 to 122, p=0.0001), and for oestrogen receptor-negative status compared with positive (OR 24.1; 95% CI 12.7 to 46.00, p=0.0001). EGFR expression may play a role in breast cancer proliferation, but appears unlikely to modify tumour pathology via postulated mechanisms of oestrogen receptor/EGFR-mediated cross-talk.

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Known collectively as serrated polyps, hyperplastic polyps (HP), sessile serrated adenomas (SSA/SSP) and traditional serrated adenoma (TSA) may represent a spectrum of increasing malignant potential with characteristic immunological markers. There is increasing evidence that HP, SSA/SSP and TSA are biologically different and are likely to represent a spectrum along the serrated polyp pathway. Although there is general consensus about the diagnostic features of serrated polyps, the morphological differences between the categories are often subtle. This study compares the expression of p53 and P504S among serrated polyps. Sixty seven randomly selected biopsies (n = 59) and resection specimens (n = 8) histologically diagnosed for SSA/SSP, TSA and HP (19, 30 and 18 specimens, respectively) were obtained.
There was a significant difference in p53 (P < 0.001) and P504S (P < 0.001) immunopositivity and distribution among the serrated polyps. In particular, there is diffuse expression p53 and P504S in TSA compared to HP and SSA/SSP where p53 and P504S expression was more frequently confined to the lower 1/3 of the crypts. In addition, percentage of cells expressing p53 and p504S expression was higher in TSA than those of HP and SSA/SSP.
Immunostains, p53 and P504S, may be useful adjuncts to morphological diagnosis of serrated polyps.

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The tyrosine kinase receptor, HER2 is a crucial prognostic marker and therapeutic target for breast cancer; however, the downstream targets and biological effectors of HER2 remain unclear. We investigated the relationship between HER2 and the transcription factor FoxM1 in breast cancer. HER2 and FoxM1 expression levels were compared in breast carcinoma cell lines, paraffin-embedded breast cancer patient samples and at the mRNA level in purified breast epithelial cells. To further examine the relationship between HER2 and FoxM1 expression, we either overexpressed or siRNA-mediated depleted endogenous HER2 in breast cancer cell lines. Additionally, a mammary epithelium-targeted HER2 (neu) transgenic mouse model was also used to assess the effect of HER2 on FoxM1 levels. Furthermore, the effect of the HER2-tyrosine kinase inhibitor lapatinib on FoxM1 in HER2 positive breast cancer cells was investigated. HER2 protein levels directly correlated with FoxM1 expression in both breast carcinoma cell lines and paraffin-embedded breast cancer patient samples. Moreover, in purified breast epithelial cells, overexpression of HER2 was associated with high levels of FoxM1 mRNA, suggesting that the upregulation of FoxM1 expression is at least partially mediated transcriptionally. Furthermore, overexpression or ablation of endogenous HER2 resulted in parallel changes in FoxM1 expression. Critically, mammary epithelium-targeted HER2 mouse tumours also resulted in increased FoxM1 expression, suggesting that HER2 directed FoxM1 expression occurs in vivo and may be a critical downstream effector of HER2-targeting therapies. Indeed, treatment of breast cancer cells with lapatinib reduced FoxM1 expression at protein, mRNA and gene promoter levels. Moreover, analysis of normal and breast cancer patient samples revealed that elevated FoxM1 expression at protein and mRNA levels correlated with breast cancer development, but not significantly with cancer progression and survival. Our results indicate that the HER2 receptor regulates the expression of the FoxM1 transcription factor, which has a role in breast cancer development.

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To validate the use of the silver-enhanced in situ hybridization (SISH) technique in assessing HER2 status of breast carcinoma in excision biopsy specimens, and to assess its reliability in determining HER2 status in core biopsy specimens.
Routinely processed paraffin sections of 65 excised breast carcinomas and 56 available preoperative core biopsy specimens from the same patients were selected from the archives for testing with the SISH technique using the automated Ventana Benchmark XT machine. For each case, two sections were used, one for the assessment of HER2 gene amplification and the other for assessment of chromosome 17. Of the 65 excision specimens tested, sections of 53 cases were also available for fluorescence in situ hybridization (FISH) examination. HER2 gene amplification was detected by SISH in 14 (21%) out of 65 excision specimens and in eight (14%) out of 56 core biopsy specimens. The results of SISH and FISH were identical in 50 (94%) out of the 53 excision cases examined by the two techniques. Two cases were SISH-, FISH+, and one case was the other way round. SISH results of core biopsy specimens and corresponding excision biopsy specimens were identical in 50 (89%) out of 56 cases. Four cases (7%) were SISH- in cores but positive in excision specimens, whereas two cases were the other way round.
The results validate the use of the SISH technique for assessing HER2 status of excised breast carcinoma tissue sections. The results are comparable to those obtained with FISH, but SISH has the advantage of having a permanent end result that can be visualized by an ordinary light microscope. There is a reasonable 89% concordance between SISH results obtained in core and excision biopsy specimens. However, it may be prudent to postpone doing SISH, if possible, until sections of the resected specimen are available, as these seem to be more reliable.

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Ser(167) was first identified as a major phosphorylation site of the estrogen receptor -alpha (ER) positive in the MCF7 breast cancer cell line. Subsequent studies have shown that Ser(167) phosphorylation is important in the regulation of ER activity and have identified p90RSK and AKT as protein kinases that phosphorylate Ser(167). The purpose of this study was to determine the importance of Ser(167) phosphorylation in breast cancer progression.
Immunohistochemical staining of primary breast cancer biopsies (n = 290) was carried out using antibodies specific for ER phosphorylated at Ser(167) and for phosphorylated p44/p42 mitogen-activated protein kinase (MAPK), phosphorylated p90RSK, and phosphorylated AKT.
In ER-positive breast cancer patients, Ser(167) phosphorylation was associated with low tumor grade (P = 0.011), lymph node negativity (P = 0.034), and relapse-free (P = 0.006) and overall (P = 0.023) survival. Further, Ser(167) phosphorylation was strongly associated with phosphorylated p90RSK (P < 0.001), previously shown to phosphorylate Ser(167) in vitro, as well as being associated with phosphorylated MAPK (P < 0.0005). The activities of both kinases also seemed to be indicative of better prognosis. There was, however, no association between HER2 positivity and Ser(167) phosphorylation nor were the activities of MAPK or p90RSK associated with HER2 status, suggesting that other cell surface receptors may be important in regulating these activities in breast cancer.
These findings show that phosphorylation at Ser(167) of ER predicts for likelihood of response of ER-positive breast cancer patients to endocrine therapies.

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We have shown previously that cutting or loading articular cartilage resulted in a fibroblast growth factor-2 (FGF-2) dependent activation of the extracellularly regulated kinase (ERK), and induction of a number of chondrocyte regulatory proteins including tissue inhibitor of metalloproteinase-1 and matrix metalloproteinases 1 and 3. An extracellular matrix-bound pool of FGF-2 was apparent, which could be liberated from the tissue by heparitinase (Vincent et al., Proc Natl Acad Sci U S A 2002;99(12):8259-64, Vincent et al., Arthritis Rheum 2004 Feb;50(2):526-33). Our objectives were to determine where FGF-2 was stored in articular cartilage, to which proteoglycan it was bound, and to elucidate its role in chondrocyte mechanotransduction.
Immunohistochemistry and confocal microscopy were used to localise FGF-2 in the tissue. In vitro binding studies were performed using IASYS surface plasmon resonance. To study the role of pericellular FGF-2 in mechanotransduction cartilage explants or articular chondrocytes encapsulated in alginate were loaded using an in house loading rig. The loading response was assessed by the activation of ERK, in the presence or absence of a specific FGFR inhibitor.
Here we have identified perlecan as the heparan sulphate proteoglycan that sequesters FGF-2 in articular cartilage. Perlecan and FGF-2 co-localised within the type VI collagen-rich pericellular matrix of porcine and human articular cartilage. Chondrocytes encapsulated in alginate were able to accumulate pericellular perlecan and FGF-2 in culture, and deliver an FGF-dependent activation of ERK when loaded.
Loading-induced ERK activation was dependent upon the presence and concentration of pericellular FGF-2, suggesting a functional role for this matrix-bound growth factor in chondrocyte mechanotransduction.

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Oestrogen receptor-alpha (ERalpha) is an important prognostic marker in breast cancer and endocrine therapies are designed to inhibit or prevent ERalpha activity. In vitro studies have indicated that phosphorylation of ERalpha, in particular on serine 118 (S118), can result in activation in a ligand-independent manner, thereby potentially contributing to resistance to endocrine agents, such as tamoxifen and aromatase inhibitors. Here we report the immunohistochemistry (IHC) of S118 phosphorylation in 301 primary breast tumour biopsies. Surprisingly, this analysis shows that S118 phosphorylation is higher in more differentiated tumours, suggesting that phosphorylation at this site is associated with a good prognosis in patients not previously treated with endocrine agents. However, we also report that S118 phosphorylation was elevated in tumour biopsies taken from patients who had relapsed following tamoxifen treatment, when compared to pre-treatment biopsies. Taken together, these data are consistent with the view that S118 phosphorylation is a feature of normal ERalpha function and that increases in levels of phosphorylation at this site may play a key role in the emergence of endocrine resistance in breast cancer.

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We report a primary retinal tumor with features of myxopapillary ependymoma. The lesion occurred in a 33-year-old man with a long history of phthisis bulbi and a more recent history of pain to the right eye. Enucleated ocular globe revealed a lesion occupying most of the retinal surface. Histologically, the retina was replaced by a tumor composed of spindle cells with fibrillary cytoplasm and round to ovoid nuclei forming fascicles, perivascular pseudorosettes, microcysts, and deposition of extracellular mucins. Calcifications, metaplastic bone, and lymphoplasmacytic inflammatory infiltrate were also seen. Tumor cells expressed GFAP and S-100 and to lesser extent carbonic anhydrase II. The immunoreaction for EMA showed diffuse granular positivity, decorated a few extracellular lumina, and highlighted intracytoplasmic lumina in a few cells. Ultrastructurally, there was accumulation of extracellular material between cells and around capillaries, long interdigitating cytoplasmic processes, extracellular lumina packed with microvilli, a few junctions evident around lumina, and some ciliary basal bodies and ciliary basal rootlets. As control cases, we also investigated expression of EMA and carbonic anhydrase II in an ocular globe with retinal gliosis and three cases of myxopapillary ependymoma of the cauda equina. The lesion described here represents the first example of retinal tumor with features of myxopapillary ependymoma. Pathologic features and particularly expression of carbonic anhydrase II suggest a derivation from intrinsic glial cells of retina otherwise known as Muller cells.

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Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis (RA), but little is understood about their citrullinated target antigens. We have detected a candidate citrullinated protein by immunoblotting lysates of monocytic and granulocytic HL-60 cells treated with peptidylarginine deiminase. In an initial screen of serum samples from four patients with RA and one control, a protein of molecular mass 47 kDa from monocytic HL-60s reacted with sera from the patients, but not with the serum from the control. Only the citrullinated form of the protein was recognised. The antigen was identified by tandem mass spectrometry as α-enolase, and the positions of nine citrulline residues in the sequence were determined. Serum samples from 52 patients with RA and 40 healthy controls were tested for presence of antibodies against citrullinated and non-citrullinated α-enolase by immunoblotting of the purified antigens. Twenty-four sera from patients with RA (46%) reacted with citrullinated α-enolase, of which seven (13%) also recognised the non-citrullinated protein. Six samples from the controls (15%) reacted with both forms. α-Enolase was detected in the RA joint, where it co-localised with citrullinated proteins. The presence of antibody together with expression of antigen within the joint implicates citrullinated α-enolase as a candidate autoantigen that could drive the chronic inflammatory response in RA.

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Some oestrogen-receptor (ER) positive breast cancers express epidermal growth factor receptor (EGFR), but whether inhibition of EGFR can suppress proliferation of breast cancer cells and ER function is not known.
In a double-blind, placebo-controlled randomised trial of 56 postmenopausal patients with ER-positive and EGFR-positive primary breast cancer, 27 women were randomly assigned to the tyrosine-kinase inhibitor of EGFR gefitinib (250 mg given orally once a day) and the aromatase inhibitor anastrozole (1 mg given orally once a day), and 29 women to gefitinib (250 mg given orally once a day) and placebo of identical appearance to anastrozole given orally once a day, all given for 4-6 weeks before surgery. Primary outcome was inhibition of tumour-cell proliferation, as measured by Ki67 antigen labelling index. Secondary outcomes were reduction in EGFR phosphorylation at Tyr 845, reduction in ER phosphorylation at Ser 118, tumour size, and toxic effects. Analyses were by intention to treat.
Patients assigned gefitinib and anastrozole had a greater reduction from pretreatment values in proliferation-related Ki67 labelling index than did those assigned gefitinib alone (mean % reduction 98.0 [95% CI 96.1-98.9] vs 92.4 [85.1-96.1]; difference between groups 5.6% [5.1-6.0], p=0.0054). Tumour size was reduced by 30-99% (partial response) in 14 of 28 patients assigned gefitinib and [corrected]in 12 of 22 assigned gefitinib, as assessed by ultrasonography. Reduction in phosphorylation of ER at Ser 118 was similar for both groups. Treatment was well tolerated and much the same for both groups.
Single-agent gefitinib and gefitinib combined with anastrozole are well-tolerated and effective treatments for reducing the size of breast tumours and levels of ER phosphorylation when given as neoadjuvant therapy.

[Show abstract][Hide abstract]ABSTRACT:
Antibodies against citrullinated proteins are highly specific for rheumatoid arthritis (RA), but little is understood about their citrullinated target antigens. We have detected a candidate citrullinated protein by immunoblotting lysates of monocytic and granulocytic HL-60 cells treated with peptidylarginine deiminase. In an initial screen of serum samples from four patients with RA and one control, a protein of molecular mass 47 kDa from monocytic HL-60s reacted with sera from the patients, but not with the serum from the control. Only the citrullinated form of the protein was recognised. The antigen was identified by tandem mass spectrometry as α-enolase, and the positions of nine citrulline residues in the sequence were determined. Serum samples from 52 patients with RA and 40 healthy controls were tested for presence of antibodies against citrullinated and non-citrullinated α-enolase by immunoblotting of the purified antigens. Twenty-four sera from patients with RA (46%) reacted with citrullinated α-enolase, of which seven (13%) also recognised the non-citrullinated protein. Six samples from the controls (15%) reacted with both forms. α-Enolase was detected in the RA joint, where it co-localised with citrullinated proteins. The presence of antibody together with expression of antigen within the joint implicates citrullinated α-enolase as a candidate autoantigen that could drive the chronic inflammatory response in RA.